The Norwalk-Like Viruses (NLV), now are called Noroviruses, are small round viruses within the calicivirus family and are important viral pathogens that cause acute gastroenteritis, the second most common illness in the United States. Norwalk disease is a mild to moderate illness that develops 1-2 days after infection by person-to-person transmission, surface contamination, or by contaminated food or water and the illness lasts for 24-60 hours. Symptoms include nausea, vomiting, diarrhea, abdominal pain and upon occasion headache and low fever. Severe illness requiring hospitalization is most unusual. Particularly large epidemic outbreaks of illness have occurred following consumption of contaminated water or uncooked food such as salad and ham, and shellfish including clams, cockles, and oysters.
NLVs are morphologically similar but genetically and antigenically diverse viruses. Genetically, NLVs belong to one of two genera, the “Norwalk-like viruses” (NLVs) and the “Sapporo-like viruses” of human caliciviruses, a subset of Caliciviridae. The NLV genus can be divided into three genogroups (I, II, and III). Each genogroup can be further divided into genetic clusters; at least 15 genetic clusters of NLVs have been identified (see Capsid protein diversity among the Norwalk-like viruses, Virus Genes 2000; 20:227-36, incorporated herein by reference.) NLVs encode a single capsid protein that self-assembles into virus-like particles (VLPs) when the recombinant capsid protein is expressed in baculovirus-infected insect cells (see Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein, Jiang at al., Journal of Virology, 66:6527-32 (1992), incorporated herein by reference). These VLPs are morphologically and immunologically indistinguishable from the authentic viruses found in human feces.
Since the molecular cloning of NLVs and the subsequent development of new diagnostic assays, NLVs have been recognized as the most important cause of non-bacterial epidemics of acute gastroenteritis in both developed and developing countries, affecting individuals of all ages. Over 90% of non-bacterial gastroenteritis outbreaks in the US are found to be associated with NLVs. The percentage of individuals in developing countries who have antibodies against NLVs at an early age is very high (as it is with poliovirus and other viruses transmitted by fecal contamination of water and foods). In the US the percentage increases gradually with age, reaching 50% in people over 18 years of age. The antibody prevalence to NLVs is estimated to be higher based on the data using new diagnostic methods than previously reports using old methods. Immunity, however, is not permanent and reinfection can occur.
Before molecular cloning of NV in 1990, several methods were developed for diagnosis of Norwalk virus that included immune electron microscopy and other immunologic methods such as radio immunoassays (RIAs) or enzyme linked immunoabsorbent assays (ELISAs). Because most of these methods use reagents from infected humans that are of limited source, they are not widely used. Since the molecular cloning of NLVs, several methods have been developed for diagnosis of NLVs, including reverse transcription-polymerase chain reaction (RT-PCR) for detection of the viral RNA and recombinant enzyme immune assay for detection of viral antigens and antibody against viral antigens. Due to the wide genetic and antigenic diversity of NLVs, these methods encountered problems of low detection sensitivity and specificity.
Although the Norwalk Virus was discovered in 1972, knowledge about the virus has remained limited because it has failed to grow in cell cultures and no suitable animal models have been found for virus cultivation. Human stool samples obtained from outbreaks and from human volunteer studies, therefore, are the only source of the virus. Still the concentration of the virus in stool is usually so low that virus detection with routine electron microscopy is not possible. Although limited studies on human volunteers showed that antibody response to NV challenge was protective against subsequent infection, other studies found that pre-existing antibodies against NV were not protective. Even more puzzling has been that some individuals with high levels of antibody against NV were more susceptible to the virus than individuals who did not have the antibody, and that 20-30% of individuals who did not have antibody against NV could not be infected by challenge with NV.
The most commonly encountered blood groups are ABO (ABH) and Lewis. (See Hematology Basic Principles and Practice, R. Hoffman, editor; Churchill Livingstone NY, N.Y., pub. 1995). The biosynthetic pathways used in forming antigens in the ABH, Lewis, P, and I blood group systems are interrelated. These oligosaccharide antigens may exist free in solution. In addition, they can be covalently attached to lipid molecules (ceramide) to form glycosphingolipids, or to polypeptides to form mucins, integral membrane glycoproteins, or soluble glycoproteins. The lycosidic linkages (i.e., the bonds between monosaccharides) are specifically catalyzed by glycosyltransferases. Some glycosyltransferases, found in all individuals, form framework structures. Other glycosyltransferases are allelically inherited and specify the synthesis of variable structures. Because of their variable inheritance and expression, the latter may form blood group antigens. As described below, antigens in the ABH, Lewis, P, and I blood group systems are synthesized on common precursor framework molecules. Competition between genetically inherited blood group-specific glycosyltransferases results in a rich mixture of antigenic molecules. In addition, a single oligosaccharide may contain several different blood group specificities. The absence of particular blood group antigens in certain individuals may result in specific antibody production after antigenic stimulation.
Although the ABH antigens are typically described as blood group antigens because of their presence on red cells, they are also found on other tissues, and may be more appropriately termed histo-blood group antigens. In blood, they exist in both a cellular form on platelets and a soluble form as blood group active glycosphingolipids coupled to plasma lipoproteins. They exist as membrane antigens on such diverse cells as vascular endothelial cells and intestinal, cervical, urothelial, pulmonary and mammary epithelial cells. Soluble forms are also found in various secretions and excretions, such as saliva, milk, urine, and feces. In some tissues, their appearance is developmentally regulated. Despite their wide distribution, genetic inheritance, developmental regulation, and importance in transfusion and transplantation, their normal physiological function, if any, remains a mystery.
To appreciate the structure and antigenicity of ABH antigens and their relationship to other blood group systems fully, it is necessary to understand the underlying biochemistry.
Early studies indicated that anti-A, anti-B, and anti-H antibodies specifically recognize epitopes composed of terminal trisaccharides or disaccharides. From these results it is possible to conclude that the A, B, and H antigens are not directly encoded by the corresponding genes, but rather the genes code for particular glycosyltransferases, commonly called the A, B, and H transferases, or equivalently, the A, B, and H enzymes. The H enzyme is a fucosyltranferase that specifically adds fucose in an (α-1→2) linkage to a terminal galactose. The A or B enzymes then add N-acetylgalactosamine or galactose, respectively, in an (α-1→3) linkage to the same terminal galactose. However, the substrate for the A or B enzymes is a terminal H antigen; these enzymes do not transfer the appropriate sugar to galactose in the absence of the (α-1→2)-linked fucose. Similarly, the H enzyme does not function if this galactose is substituted with a different sugar.
The finding that the A and B genes code for glycosyltransferases explains some results obtained from classic genetic analysis of family pedigrees. In particular, the A and B genes are inherited in a strict mendelian fashion and are dominant compared to O, but the A and B genes are co-dominant with each other. That is, an individual with the genotype AO (or BO) is phenotypically A (or B), an individual of genotype OO is phenotypically group O. Since the A and B enzymes both use the H antigen as substrate, even the presence of only approximately 50% of these enzymes in and AO (or BO) heterozygote is sufficient to convert the red cells to the corresponding A (or B) phenotype. Similarly, if both the A and B enzymes are present, they each convert approximately 50% of the available H antigen substrate, yielding red cells expressing both antigens A and B.
The ABH antigens are found not only on cells but also in secretions, particularly saliva and plasma. The ability to secrete ABH is genetically inherited: approximately 80% of whites are secretors and 20% are nonsecretors. This trait is inherited as a single locus gene (FUT2) in simple mendelian fashion. The secretor gene (Se) is dominant; nonsecretor (se) is recessive. The terminal carbohydrate sequences of the ABH antigens in saliva and plasma are identical to those on red cells. At least one copy of the Se gene is found in approximately 80% of the population and leads to the expression of ABH antigens in secretions. By contrast, the traditional H locus is a structural gene called FUT1. This gene is active in virtually all individuals, with rare defective mutation such as the “Bombay” blood type, and leads to the formation of ABH antigens on red blood cells and other tissues.
The two Lewis blood group antigens Lea (Lewis a) and Leb (Lewis b) were discovered in the 1940s. Virtually all individuals fall into one of three different Lewis types Le(a+b−), Le(a−b+), and Le(a−b−). A type of Le(a+b+) is seen among Asian populations. These molecules are not intrinsic red blood cell antigens; they are synthesized in another tissue (probably the intestinal epithelium), circulate in plasma attached to lipoproteins, and then passively transfer onto red cells. Biochemical studies have demonstrated that these are carbohydrate antigens on glycosphingolipids. They are structurally similar to the type ABH antigens found on plasma glycosphingolipids that likewise transfer onto red blood cells. The Lewis gene codes for an enzyme, an (α-1→4) fucosyltransferase, and thus behaves in a dominant fashion. The transfer of fucose to a type 1 precursor by the Lewis enzyme results in the formation of the Lea antigen; the addition of (α-1→4) linked fucose to the H type 1 structure leads to the formation of the Leb antigen. Thus the Leb antigen is formed through the cooperation of two glycosyltransferases encoded by two genes, one gene for the Lewis system (Le) and one from the ABH system (Se) or, equivalently, H type 1 at a different, unlinked locus, demonstrating the connections of the ABH, Secretor, and Lewis systems. Since the secretor enzyme converts virtually all type 1 precursor into H type 1, whether or not the Lewis enzyme is present, Lewis-positive secretors have virtually no Lea antigen, and their red blood cells type as Le(a−b+). By contrast Lewis-positive nonsecretors have Le(a+b−) red blood cells.
Histo-blood group antigens have been linked to infection by several bacterial and viral pathogens. This suggests that the histo-blood group antigens are a recognition target for pathogens and may facilitate entry into a cell that expresses or forms a receptor-ligand bond with the antigens. While the exact nature of such an interaction is unknown, close association of a pathogen that would occur with antigen binding may play a role in anchoring the pathogen to the cell as an initial step in the infection process. Interactions of some parasites and bacteria with human cells have been shown to depend on the presence of certain blood group antigens. For example, P. vivax malarial parasites only enter human red blood cells when the Fy6 Duffy blood group protein is present on the cells. Certain E. coli will only attach to the epithelial cells of the urinary tract if P or Dr blood group antigens are present in the epithelial cells. The P antigen is also the red blood cell receptor for Parvovirus B19. Leb antigen has recently been found to be the receptor for H. pylori in the gastric tissue. The high frequency blood group antigen known as AnWj, is the red blood cell receptor for H. influenzae. Since the relevance of ABH blood group antigens as parasitic/bacterial/viral receptors and their association with immunologically important proteins is now well established, the prime biologic role for ABH blood group antigens may well be independent and unrelated to the erythrocyte.
A recent study has shown that a relationship may exist between a person's ABO histo-blood group type and the risk of an infection and symptomatic disease after clinical challenge by the Norwalk Virus (NV). (See Norwalk Virus Infection and Disease is Associated with ABO-Histo-Blood Group Type, The Journal of Infectious Diseases, 185:1335-7 (2002), incorporated herein by reference). The study shows that persons of blood type O were significantly more likely to become infected with the NV, while persons of blood type B and AB had a decreased risk of infection, and that blood type B persons did not develop symptomatic illnesses despite being challenged with the NV.
Despite the advances made in recognizing that human histo-blood type may affect the risk of infection by the Norwalk Virus, there has been no explanation of the specific binding mechanism used by NLVs to infect human epithelium cells in the gastrointestinal (GI) tract, and no explanation of the specific binding relationships between NLVs, including the NV, and the human histo-blood group antigens. Furthermore, there has not been shown an effective means to treat a NLV infection and/or its illness.
Therefore, the need has remained to understand: the specific mechanism for NLV infection within the GI tract, the specific binding properties of the prototype NV with the ABO blood antigens and the Le blood antigens, the specific binding properties of the other NLVs with the human histo-blood phenotypes and their respective blood antigens, and the compounds and compositions that are effective to inhibit binding between NLVs and blood antigens, to prevent or treat an infection by a NLV and/or the resulting illness.